Obesity is widely recognized as a serious health problem for the developed countries, and has reached epidemic status in the United States. More than 50% of the U.S. population is considered overweight, with  greater than 25% diagnosed as clinically obese and at considerable risk for heart disease, non-insulin dependent diabetes mellitus (NIDDM), hypertension, and certain cancers. This epidemic presents a significant burden on the health care system as projected obesity treatment costs of more than $70 billion annually are expected in the U.S. alone. Strategies for treating obesity include reducing food intake or enhancing the expenditure of energy.
It has been demonstrated that, when injected into the third ventricle of the brain or intraperitoneally, a cyclic heptapeptide analog of xcex1-melanocyte stimulating hormone (xcex1MSH) having melanocortin-4 receptor (MC4-R) agonist activity caused long lasting inhibition of food intake in mice. This effect was reversible when co-administered with a MC4-R antagonist. (Fan, et al., Nature (1997) 385: 165-168) Therefore, agonists of MC4-R activity would be useful in treating or preventing obesity.
There are five known melanocortin receptors based on sequence homology that ranges from 35-60% homology between family members ((Cone, et al., Rec. Prog. Hormone Res. (1996) 51: 287-318), but these receptors differ in their functions. For example, the MC1-R is a G-protein coupled receptor that regulates pigmentation in response to the xcex1MSH, which is a potent agonist of MC1-R. (Cone, et al., ibid.). Agonism of the MC1-R receptor results in stimulation of the melanocytes which causes eumelanin and increases the risk for cancer of the skin. Agonism of MC1-R can also have neurological effects. Stimulation of MC2-R activity can result in carcinoma of adrenal tissue. The effects of agonism of the MC3-R and MC5-R are not yet known. All of the melanocortin receptors respond to the peptide hormone class of melanocyte stimulating hormones (MSH). These peptides are derived from pro-opiomelanocortin (POMC), a prohormone of 131 amino acids that is processed into three classes of hormones; the melanocortins (xcex1, xcex2 and xcex3), adrenocorticotropin hormone (ACTH), and various endorphins (e.g. lipotropin) (Cone, et al., ibid.). Because of their different functions, simultaneous agonism of the activities of multiple melanocortin receptors has the potential of causing unwanted side effects. Therefore it is desirable that an agonist of MC4-R be more selective for the MC4-R than for one or more of the other melanocortin receptors.
Haskell-Luevano, et al. (Peptides (1996) 17(6): 995-1002) disclose peptides that contain the tripeptide (D)Phe-Arg-Trp and exhibit melanotropic (skin darkening) activity in the frog (Rana pipiens) skin bioassay. Haskell-Luevano, et al. (ibid.) do not disclose any compound of formula I, II or III described below.
This invention provides a compound of the formula: 
In compounds of formula I m is 0 or 1. n is 0 or 1. R1 is an unsubstituted linear or branched alkyl having from 1 to 8 carbon atoms; linear or branched alkyl having from 1 to 8 carbon atoms mono-substituted by phenyl or carboxyl; unsubstituted phenyl; or phenyl mono-substituted by fluoro, chloro or linear or branched alkyl having from 1 to 4 carbon atoms. X is 
R2, R3 and R4 are independently hydrogen or a linear or branched alkoxy having from 1 to 4 carbon atoms, wherein when R3 is alkoxy, R2 and R4 are both hydrogen. R9 is hydrogen, linear or branched alkyl having from 1 to 3 carbons, linear or branched alkoxy having from 1 to 3 carbons, or unsubstituted phenoxy. R11 is cyclohexyl, cycloheptyl, or a branched alkyl having from 3 to 8 carbon atoms. R6 is hydrogen or methyl. R7 is 
and R8 is hydrogen or methyl; or
Y is 
and R8 is hydrogen.
This invention provides a compound of the formula: 
In the compounds of formula II m is 0 or 1. n is 0 or 1. R1 is an unsubstituted linear or branched alkyl having from 4 to 8 carbon atoms; linear or branched alkyl having from 1 to 8 carbon atoms mono-substituted by phenyl or carboxyl; or unsubstituted phenyl; or phenyl mono-substituted by fluoro, chloro or linear or branched alkyl having from 1 to 4 carbon atoms. R7 is 
and R8 is hydrogen or methyl; or
Y is 
and R8 is hydrogen.
R10 is hydrogen, halo, linear or branched alkyl having from 1 to 3 carbon atoms, linear or branched alkoxy having from 1 to 3 carbon atoms, or xe2x80x94NR12N13 wherein R12 and R13 are each independently a linear or branched alkyl having from 1 to 3 carbons or together are xe2x80x94(CH2)qxe2x80x94 wherein q is 3, 4 or 5.
This invention provides a compound of the formula: 
In the compounds of formula III, R1 is unsubstituted linear or branched alkyl having from 4 to 8 carbon atoms. R6 is hydrogen or methyl. R8 is hydrogen or methyl. p is 2, 3 or 4 and R14 is 
or p is 4 and R14 is 
or p is 3 and R14 is 
The compounds of formulae I, II and III as well as Penta-Adpc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-Ape-(D)Phe-Arg-Trp-Gly-NH2 are agonists of the MC4-R. It is known that agonists of MC4-R activity cause reduction of food intake in a mouse model of human obesity. Therefore the compounds of formula I are useful in the treatment or prevention of obesity.
All of the compounds of formulae I, II and III exemplified below as well as Penta-Adpc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-Ape-(D)Phe-Arg-Trp-Gly-NH2 were tested for MC4-R agonist activity and MC1-R agonist activity in the in vitro assay described below in Biological Activity Example A. All of the tested compounds had an EC50 for MC4-R agonist activity of less than 500 nM, and all exhibited at least 10-fold greater MC4-R agonist activity than MC1-R agonist activity. In contrast, the compound Bu-His-(D)Phe-Arg-Trp-Gly-NH2 (Example 30) exhibited greater MC1-R agonist activity than MC4-R agonist activity.
The nomenclature used to define the peptides is that typically used in the art wherein the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus appears to the right. By natural amino acids is meant one of the naturally occurring amino acids found in proteins, i.e., Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp, and His. Where the amino acid has isomeric forms, it is the L form of the amino acid that is represented unless otherwise explicitly indicated.
The following abbreviations or symbols are used to represent amino acids, protecting groups, solvents, reagents and the like.
Setting forth the substituted amino acid, in parentheses indicates analogs of the peptide sequence. Derivatization of the N-terminal amino group, is indicated to the left of the N-terminal substitution, separated by a hyphen. That is, for example, Ac-His-(D)Phe-Arg-Trp-Gly-NH2 indicates a peptide having an amino acid sequence in which an acetyl group has been substituted for hydrogen at the N-terminus. The suffixes xe2x80x9cxe2x80x94OHxe2x80x9d and xe2x80x9cxe2x80x94NH2xe2x80x9d following the hyphen or the parentheses refer to the free acid and amide forms of the polypeptide, respectively.
In compounds of formula I, it is generally preferred that R6 and R8 are both hydrogen, n is 1 and R7 is either the first or the second of the substructures shown above. Also preferred are compounds of formuae IA, IB or IC as shown below.
Compounds of formula IA, are represented as follows: 
In the compound of formula IA, m is 0 or 1. n is 0 or 1. R1 is an unsubstituted linear or branched alkyl having from 1 to 8 carbon atoms; linear or branched alkyl having from 1 to 8 carbon atoms mono-substituted by phenyl or carboxyl; unsubstituted phenyl; or phenyl mono-substituted by fluoro, chloro or linear or branched alkyl having from 1 to 4 carbon atoms. R2, R3 and R4 are independently hydrogen; a linear or branched alkyl having from 1 to 4 carbon atoms; hydroxy, a linear or branched alkoxy having from 1 to 4 carbon atoms; or chloro, wherein when R3 is alkyl, hydroxy, alkoxy or chloro, R2 and R4 are both hydrogen. R6 is hydrogen or methyl. R7 is 
and R8 is hydrogen or methyl; or
Y is 
R8 is hydrogen.
In the compounds of formula IA, R7 can be either a tryptophan side chain or a 1- or 2-naphthyl group. In compounds of formula IA in which R7 is a tryptophan side chain, i.e. 
n can be either 0 or 1. Examples of such compounds in which n is 0 include Penta-Apc-(D)Phe-Arg-Trp-NH2 and Penta-Apc-(D)Phe-Arg-N-methylTrp-NH2. In compounds of formula IA in which R7 is a tryptophan side chain and n is 1, Y can be a linear or branched alkyl group selected from methylene, ethylene or methyl-substituted methylene, i.e. 
or one of the aryl-containing moieties shown above. In compounds of formula IA in which R7 is a tryptophan side chain and n is 1, Y is methylene, ethylene or methyl-substituted methylene, m can be 0 or 1. Examples of such compounds in which m is 1 include Bu-Carbamoyl-Apc-(D)Phe-Arg-Trp-Gly-NH2, Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-Ala-NH2, and Bu-Carbamoyl-Apc-(D)Phe-Arg-Trp-xcex2-Ala-NH2. In compounds of formula IA in which R7 is a tryptophan side chain, n is 1, Y is methylene, ethylene or methyl-substituted methylene and m is 0, the phenyl ring of the Apc group can be either unsubstituted (i.e. R2, R3 and R4 are hydrogen) or substituted. In such compounds in which the phenyl ring of the Apc group is unsubstituted, R1 can be, for example, an unsubstituted linear alkyl such as in the compounds Penta-Apc-(D)Phe-Arg-Trp-Gly-NH2, Penta-Apc-(D)Phe-Arg-Trp-Sar-NH2, Penta-Apc-(D)Phe-Arg-N-methylTrp-Gly-NH2, Bu-Apc-(D)Phe-Arg-Trp-Ala-NH2, or Bu-Apc-(D)Phe-Arg-Trp-xcex2-Ala-NH2; or unsubstituted phenyl such as in the compounds Phenylacetyl-Apc-(D)Phe-Arg-Trp-Gly-NH2, Phenylacetyl-Apc-(D)Phe-Arg-Trp-Ala-NH2, or Phenylacetyl-Apc-(D)Phe-Arg-Trp-Ala-NH2. In such compounds in which the phenyl ring of the Apc group is substituted, one preferred substitution pattern is wherein R3 is alkyl, hydroxy, alkoxy or chloro (more preferably R3 is hydroxy or alkoxy) and R2 and R4 are hydrogen. Examples include Penta-4-ClApc-(D)Phe-Arg-Trp-Gly-NH2, Penta-4-MeApc-(D)Phe-Arg-Trp-Gly-NH2, Penta-4-HOApc-(D)Phe-Arg-Trp-Gly-NH2, Penta-4-MeOApc-(D)Phe-Arg-Trp-Gly-NH2, Penta-4-EtOApc-(D)Phe-Arg-Trp-Gly-NH2, and Penta-4-iPrOApc-(D)Phe-Arg-Trp-Gly-NH2. Another preferred substitution pattern is wherein R2 is alkoxy, R3 is hydrogen and R4 is hydrogen, for example in the compound Penta-3-MeOApc-(D)Phe-Arg-Trp-Gly-NH2. In compounds of formula IA in which R7 is a tryptophan side chain and n is 1, and Y is 
m can be 0 or 1. Examples of such compounds in which m is 1 include Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-2-Aba-NH2 and Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-3-Amb-NH2. Examples of such compounds in which m is 0 include Bu-Apc-(D)Phe-Arg-Trp-2-Aba-NH2, Phenylacetyl-Apc-(D)Phe-Arg-Trp-2-Aba-NH2, Bu-Apc-(D)Phe-Arg-Trp-3-Amb-NH2, Phenylacetyl-Apc-(D)Phe-Arg-Trp-3-Amb-NH2, Bu-Apc-(D)Phe-Arg-Trp-4-Amb-NH2, and Phenylacetyl-Apc-(D)Phe-Arg-Trp-4-Amb-NH2.
In compounds of formula IA in which R7 is 2-naphthyl, i.e. 
it is preferred that R2, R3 and R4 are hydrogen. Examples of such compounds include Penta-Apc-(D)Phe-Arg-N-methyl(2)Nal-NH2 and Bu-Carbamoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH2. In compounds of formula IA in which R7 is 2-naphthyl, it is preferred that n is 1 and m is 0. In compounds of formula IA in which R7 is 2-naphthyl, n is 1 and m is 0, and Y is methylene, ethylene or methyl-substituted methylene, R1 can be, for example an unsubstituted linear alkyl. Examples of such compounds include, Penta-Apc-(D)Phe-Arg-(2)Nal-Gly-NH2, Bu-Apc-(D)Phe-Arg-(2)Nal-Gly-NH2, Ac-Apc-(D)Phe-Arg-(2)Nal-Gly-NH2, Penta-Apc-(D)Phe-Arg-N-methyl (2)Nal-Gly-NH2, Bu-Apc-(D)Phe-Arg-(2)Nal-Ala-NH2, and Bu-Apc-(D)Phe-Arg-(2)Nal-beta-Ala-NH2. Alternatively R1 can be, for example, unsubstituted phenyl, or alkyl substituted by phenyl or carboxyl. Examples of such compounds include Benzoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH2, 3-carboxylpropanoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH2, and 3-carboxylpropanoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH2. In compounds of formula IA in which R7 is 2-naphthyl, n is 1 and m is 0, and Y is 
It is preferred that R1 is unsubstituted lower alkyl. Examples of such compounds include Bu-Apc-(D)Phe-Arg-(2)Nal-3-Amb-NH2, Bu-Apc-(D)Phe-Arg-(2)Nal-2-Aba-NH2, and Bu-Apc-(D)Phe-Arg-(2)Nal-4-Amb-NH2.
Compounds of formula IB are represented as follows: 
In the compound of formula IB, R1 is an unsubstituted linear or branched alkyl having from 1 to 8 carbon atoms. R7 is 
R11 is cyclohexyl, or a branched alkyl having from 3 to 8 carbon atoms. Y is methylene, i.e. xe2x80x94CH2xe2x80x94. Examples of compounds of formula IB include Penta-Abc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-Achc-(D)Phe-Arg-Trp-Gly-NH2.
Compounds of formula IC are represented as follows: 
In the compound of formula IC, R1 is an unsubstituted linear or branched alkyl having from 1 to 8 carbon atoms. R7 is 
R9 is hydrogen, a linear or branched alkyl having from 1 to 3 carbon atoms, a linear or branched alkoxy having from 1 to 3 carbon atoms, fluoro, chloro, or unsubstituted phenoxy. Examples of compounds of formula IC in which R9 is hydrogen include Penta-Appc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-Appc-(D)Phe-Arg-(2)Nal-Gly-NH2. Examples of compounds of formula IC in which R9 is a linear or branched alkyl having from 1 to 3 carbon atoms include Penta-2-MeAppc-(D)Phe-Arg-Trp-Gly-NH2, Penta-2-iPrAppc-(D)Phe-Arg-Trp-Gly-NH2, Penta-3-MeAppc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-4-MeAppc-(D)Phe-Arg-Trp-Gly-NH2. Examples of compounds of formula IC in which R9 is a linear or branched alkoxy having from 1 to 3 carbon atoms or unsubstituted phenoxy include Penta-3-MeOAppc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-4-PhOAppc-(D)Phe-Arg-Trp-Gly-NH2. Examples of compounds of formula IC in which R9 is chloro include Penta-4-ClAppc-(D)Phe-Arg-Trp-Gly-NH2.
In the compound of formula II it is generally preferred that R6 and R8 are hydrogen. R7 can be, for example a tryptophan side chain, i.e. 
or 2-naphthyl. When R7 is a tryptophan side chain it is generally preferred that n is 1. Among the compounds of formula II in which R6 and R8 are hydrogen; R7 is a tryptophan side chain, and n is 1, are included compounds in which Y is xe2x80x94CH2xe2x80x94 and m is 0. Examples of such compounds in which R10 is hydrogen or a linear or branched alkyl having from 1 to 3 carbon atoms are included Bu-Atc-(D)Phe-Arg-Trp-Gly-NH2, Penta-5-Me-(D,L)Atc-(D)Phe-Arg-Trp-Gly xe2x80x94NH2, Penta-5-Et-(D,L)Atc-(D)Phe-Arg-Trp-Gly -NH2 and Penta-5-iPr-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH2. Examples of such compounds in which R10 is halo include Penta-5-Br-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH2, Penta-5-Br-Atc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-5-Cl-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH2. Examples of such compounds in which R10 is linear or branched alkoxy having from 1 to 3 carbon atoms include Penta-5-MeO-(D,L)Atc-(D)Phe-Arg-Trp-Gly xe2x80x94NH2, Penta-5-EtO-(D,L)Atc-(D)Phe-Arg-Trp-Gly xe2x80x94NH2 and Penta-5-iPrO-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH2. Examples of such compounds in which R10 is xe2x80x94NR12R13 wherein R2 and R13 are each methyl include Penta-5-DmaAtc-(D)Phe-Arg-Trp-Gly-NH2.
Among the compounds of formula II in which R6 and R8 are hydrogen; R7 is a tryptophan side chain, and n is 1, are included compounds in which Y is 
and R10 is halo. Examples of such compounds include Bu-(D,L)5-BrAtc-(D)Phe-Arg-Trp-2-Aba-NH2, Bu-carbamoyl-(D,L)-5-BrAtc-(D)Phe-Arg-Trp-2-Aba-NH2 and Phenylacetyl-(D,L)-5-BrAtc-(D)Phe-Arg-Trp-2-Aba-NH2.
In compounds of formula II in which wherein R6 and R8 are hydrogen; R7 is 2-naphthyl i.e. 
it is generally preferred that R10 is halo. Examples of such compounds include Penta-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH2, 3-carboxylpropanoyl-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH2, Phenylacetyl-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH2 and Bu-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-2-Aba-NH2.
Examples of compounds of formula III include Bu-Apc-(D)Phe-PhenylhomoArg-Trp-Gly-NH2, Penta-Apc-(D)Phe-Cit-Trp-Gly-NH2, Penta-Adpc-(D)Phe-Arg-Trp-Gly-NH2 and Penta-Ape-(D)Phe-Arg-Tip-Gly-NH2.
The compounds of this invention can be readily synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids. Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acid or residue thereof having its carboxyl group or other reactive groups protected and the free primary carboxyl group of another amino acid or residue thereof having its amino group or other reactive groups protected.
The synthesis of these compounds may be carried out by a procedure whereby each amino acid in the desired sequence is added one at a time in succession to another amino acid or residue thereof or by a procedure whereby peptide fragments with the desired amino acid sequence are first synthesized conventionally and then condensed to provide the desired peptide.
Such conventional procedures for synthesizing the novel compounds of the present invention include for example any solid phase peptide synthesis method. In such a method the synthesis of the novel compounds can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods [Merrifield, R. B., J. Amer. Chem. Soc. 1963, 85, 2149-2154; Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980)].
Common to chemical syntheses of peptides is the protection of reactive side chain groups of the various amino acid moieties with suitable protecting groups, which will prevent a chemical reaction from occurring at that site until the protecting group is ultimately removed. Usually also common is the protection of the alpha amino group of an amino acid or fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha amino protecting group and allow a subsequent reaction to take place at that site. While specific protecting groups are mentioned below in regard to the solid phase synthesis method, it should be noted that each amino acid can be protected by any protective group conventionally used for the respective amino acid in solution phase synthesis.
For example, alpha amino groups may be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl. Herein, Fmoc is the most preferred for alpha amino protection.
Guanidino groups may be protected by a suitable protecting group selected from nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, and Boc. Pmc is the most preferred for arginine (Arg).
In the examples all solvents, isopropanol (iPrOH), methylene chloride (CH2Cl2), dimethylformamide (DMF) and N-methylpyrrolidinone (NMP) were purchased from Fisher or Burdick and Jackson and were used without additional distillation. Trifluoroacetic acid was purchased from Halocarbon or Fluka and used without further purification. Diisopropylcarbodiimide (DIC) and diisopropylethylamine (DIPEA) was purchased from Fluka or Aldrich and used without further purification. Hydroxybenzotriazole (HOBT) dimethylsulfide (DMS) and 1,2-ethanedithiol (EDT) were purchased from Sigma Chemical Co. and used without further purification. Protected amino acids were generally of the L configuration and were obtained commercially from Bachem, Advanced ChemTech, or Neosystem. Purity of these reagents was confirmed by thin layer chromatography, NMR and melting point prior to use. Benzhydrylamine resin (BHA) was a copolymer of styrenexe2x80x941% divinylbenzene (100-200 or 200-400 mesh) obtained from Bachem or Advanced Chemtech. Total nitrogen content of these resins were generally between 0.3-1.2 meq/g.
High performance liquid chromatography (HPLC) was conducted on a LDC apparatus consisting of Constametric I and III pumps, a Gradient Master solvent programmer and mixer, and a Spectromonitor III variable wavelength UV detector. Analytical HPLC was performed in reversed phase mode using Vydac Clg columns (0.4xc3x9730 cm). Preparative HPLC separations were run on Vydac columns (2xc3x9725 cm).
Peptides were prepared using solid phase synthesis following the principles and general method described by Merrifield, [J. Amer. Chem. Soc., 1963, 85, 2149], although other equivalent chemical synthesis known in the art could be used as previously mentioned. Solid phase synthesis is commenced from the C-terminal end of the peptide by coupling a protected alpha-amino acid to a suitable resin. Such a starting material can be prepared by attaching an alpha-amino-protected amino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, or by an amide bond between an Fmoc-Linker, such as p-[(R,S)-xcex1-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyacetic acid (Rink linker) to a benzhydrylamine (BHA) resin. Preparation of the hydroxymethyl resin is well known in the art. Fmoc-Linker-BHA resin supports are commercially available and generally used when the desired peptide being synthesized has an unsubstituted amide at the C-terminus.
In general, the amino acids or mimetics are coupled onto the Fmoc-Linker-BHA resin using the Fmoc protected form of amino acid or mimetic, with 2-5 equivalents of amino acid and a suitable coupling reagent. After couplings, the resin may be washed and dried under vacuum. Loading of the amino acid onto the resin may be determined by amino acid analysis of an aliquot of Fmoc-amino acid resin or by determination of Fmoc groups by UV analysis. Any unreacted amino groups may be capped by reacting the resin with acetic anhydride and diisopropylethylamine in methylene chloride.
The resins are carried through several repetitive cycles to add amino acids sequentially. The alpha amino Fmoc protecting groups are removed under basic conditions. Piperidine, piperazine or morpholine (20-40% v/v) in DMF may be used for this purpose. Preferably 40% piperidine in DMF is utilized
Following the removal of the alpha amino protecting group, the subsequent protected amino acids are coupled stepwise in the desired order to obtain an intermediate, protected peptide-resin. The activating reagents used for coupling of the amino acids in the solid phase synthesis of the peptides are well known in the art. For example, appropriate reagents for such syntheses are benzotriazol-1-yloxy-tri-(dimethylamino) phosphonium hexafluorophosphate (BOP), Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylcarbodiimide (DIC). Preferred here are HBTU and DIC. Other activating agents as described by Barany and Merrifield [The Peptides, Vol. 2, J. Meienhofer, ed., Academic Press, 1979, pp 1-284] may be utilized. Various reagents such as 1-hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (HOSu) and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBT) may be added to the coupling mixtures in order to optimize the synthetic cycles. Preferred here is HOBT.
The protocol for a typical synthetic cycle is as follows:
Protocol 1
Solvents for all washings and couplings were measured to volumes of 10-20 ml/g resins. Coupling reactions throughout the synthesis were monitored by the Kaiser ninhydrin test to determine extent of completion [Kaiser et at. Anal. Biochem. 1970, 34, 595-598]. Slow reaction kinetics was observed for Fmoc-Arg (Pmc) and for couplings to secondary amines by sterically hindered acids. Any incomplete coupling reactions were either recoupled with freshly prepared activated amino acid or capped by treating the peptide resin with acetic anhydride as described above. The fully assembled peptide-resins were dried in vacuum for several hours.
For each compound, the blocking groups were removed and the peptide cleaved from the resin by the following procedure. Generally, the peptide-resins were treated with 100 xcexcL ethanedithiol, 100 xcexcL dimethylsulfide, 300 xcexcL anisole, and 9.5 mL trifluoroacetic acid, per gram of resin, at room temperature for 120 min. The resin is filtered off and the filtrates are precipitated in chilled ethyl ether. The precipitates are centrifuged and the ether layer is decanted. The residue was washed with two or three volumes of Et2O and recentrifuged. The crude products are dried under vacuum.
Purification of the crude peptides was carried out by preparative HPLC. The peptides were applied to the columns in a minimum volume of either AcOH/H2O or 0.1% TFA/H2O. Gradient elution was generally started at 10% B buffer, 10%-60% B in 90 minutes, (buffer A: 0.1% TFA/H2O, buffer B:0.1% TFA/CH3CN) at a flow rate of 8 mL/min. UV detection was made at 280 nm. Fractions were collected at 1.0-2.5 minute intervals and inspected by analytical HPLC. Fractions judged to be of high purity were pooled and lyophilized.
Purity of the final products was checked by analytical HPLC on a reversed phase column as stated above. Purity of all products was judged to be approximately 95-99%. All final products were also subjected to fast atom bombardment mass spectrometry (FAB-MS) or electrospray mass spectrometry (ES-MS). All products yielded the expected parent M+H ions within acceptable limits.
Utilizing the techniques described above, the compounds of this invention can be synthesized in accordance with the following reaction schemes. 
The synthetic peptides of the current invention are prepared by using conventional solid phase peptide synthesis methodology discussed in the previous section. Each cycle consists of two procedures; the initial cleavage of the Fmoc protecting group from the terminal nitrogen in the resin bound chain followed by acylation of the amine function with an Fmoc protected amino acid. The cycle is generally carried out in accordance with the stepwsize procedures outlined in Protocol 1. The deprotection is accomplished by using an organic base, for example piperazine, morpholine or piperidine, preferably piperidine in a suitable inert solvent, for example N,N-dimethylformamide (DMF) or Nmethylpyrrolidone (NMP). The coupling reaction can be carried out by one of the many conditions developed for amide bond formation, for example O-benzotriazol-l-yl N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate (HBTU) in the presence of an organic base, for example diisopropylethylamine (DIPEA) in an inert solvent, for example DMF. Alternatively in the present instance, the amide group can be formed using a carbodiimide, for example, diisopropylcarbodiimide (DIC) along with an activating agent such as 1-hydroxybenzotriazole (HOBT) in a suitable inert solvent such as DMF.
In Scheme A, in the first cycle, the Fmoc-Linker-BHA Resin represented by structure 1 is deprotected and condensed with Fmoc-amino acids of structure 2 to give the resin bound compounds of structure 3. A second cycle incorporates the Fmoc-amino acids 4 to give the compounds of structure 5 (n=1). Compounds of structure 5 in which n=0 are prepared by eliminating the first cycle, and by coupling Fmoc-amino acids of structure 4 directly to the deprotected Fmoc-Linker-BHA Resin. In the third cycle, treatment of the resin linked peptide furnishes the intermediates of structure 6a where R6 represents hydrogen. The intermediates of structure 6b where R6 represents methyl are synthesized as shown in Scheme C. Compounds of structure 6a, prepared by treating compounds of structure 5 as prescribed in steps 1-5 of Protocol 1, are reacted with an arylsulfonyl chloride, preferably 2-nitrobenzenesulfonyl chloride. The reaction is carried out in the presence of a proton acceptor, for example pyridine, triethylamine (TEA) or DIPEA, preferably DIPEA in a suitable inert solvent, preferably DMF. N-methylation of the formed sulfonamide group in the washed resin bound compounds of structure 19 is accomplished under Mitsunobu conditions. Thus the sulfonamides of structure 19 are reacted with methanol in the presence of diethyl azodicarboxylate (DEAD) and triphenylphosphine using methanol as solvent. After the reaction is complete, the resin bound N-methylsulfonamide of structure 20 is washed free of residual reagents and byproducts. The 2-nitrobenzenesulfonyl residue is removed by reacting 20 with 2-mercaptoethanol and the strong organic base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in a suitable solvent, preferably DMF to give the resin bound intermediate of structure 6b. The third cycle is completed by coupling compounds of either structures 6a and 6b with Fmoc-Arg(Pmc)-OH (7) to give the resin bound compounds of structure 8. Two additional cycles (Scheme B) are carried out on peptides of structure 8 where the amino acid Fmoc-(D)-Phe-OH (9) followed by either one of the amino acid derivatives of structure 10 or 11 are sequentially incorporated into the resin bound peptide to give the resin bound polypeptides of structures 12 and 13.
Removal of the Fmoc from the resin bound polypeptides 12 is carried out by treatment of 12 with piperidine in DMF to give the compounds of structure 14 using the reaction conditions outlined in Steps 1-5 of Protocol 1. The polypeptide is then N-capped by reaction with an acylating agent to form the resin bound amides of structure 15 or by reaction with an isocyanate to form the ureas of structure 16 (Scheme D). The acylation is carried out under a variety of methods well known to one skilled in the art. Among the methods used are:
(i) reaction of the compounds of structure 14 with a carboxylic acid R1xe2x80x94CO2H in a suitable solvent, such as DMF in the presence of HBTU, and an organic base, preferably DIPEA and
(ii) reaction of the compounds of structure 14 with a carboxylic acid chloride R1xe2x80x94COCl in a suitable solvent, such as dichloromethane in the presence an organic base, such as pyridine, TEA and DIPEA, preferably DIPEA and
(iii) reaction of the compounds of structure 14 with a carboxylic acid anhydride (R1xe2x80x94CO2COxe2x80x94R1 in a suitable solvent, such as dichloromethane or DMF in the presence an organic base, preferably DIPEA.
The reaction of the compounds of structure 14 with an isocyanate R1xe2x80x94NCO is carried out in a suitable solvent, such as dichloromethane or DMF in the presence an organic base, preferably DIPEA.
When the acylation and urea forming reactions are complete, the resin bound products 15 and 16 are washed free of residual reagents and byproducts.
Using the same conditions, the resin bound polypeptides of structure 13 are converted to the N-acylated compounds of structure 17 and the ureas of structure 18 (Scheme E).
In Scheme F, the sequencing is carried out as in Scheme A except that Fmoc-Glu(allyl)-OH (21) is incorporated into the resin bound polypeptide instead of Fmoc-Arg(Pmc)-OH (7) to give the resin bound N-capped polypeptides of structure 22. The allyl group is removed by treatment of 22 with tributyltin hydride, palladium chloride and triphenylphosphine in an inert solvent, for example DMF to give the resin bound polypeptide of structure 23. Coupling of 23 with Boc-guanidine gave the acylguanidine resin bound compounds of structure 24. The reaction can be carried out by using standard amide forming reaction methods, for example in the presence of HBTU and an organic base, preferably DIPEA in a suitable solvent, such as DMF.
In Scheme G, the sequencing is carried out as in Scheme A except that either FmocPhenylhomoArg-OH (25) or Fmoc-citrulline (26) is incorporated into the resin bound polypeptide in the place of Fmoc-Arg(Pmc)-OH (7) to give the resin bound N-capped polypeptides of structures 27 and 28 respectively.
As shown in Scheme H, the cleavage of remaining protecting groups in the N-capped polypeptides 15-18, 24, 27 and 28 and the concomitant cleavage of the peptides from the solid support is carried out by using a strong organic acid, preferably trifluoroacetic acid, optionally in the presence of an inert solvent such as dichloromethane and a trace (1%) of water. The reaction is conveniently carried out with or without the presence of one or more carbocation scavengers, for example ethanedithiol, dimethyl sulfide, triethylsilane and anisole. The polypeptide cleavage solution is filtered free from the solid support, then is diluted with a suitable solvent, preferably diethyl ether. The solid polypeptides of structures 29-35 produced in this manner is purified by reversed phase chromatography over a preparative C18 column. If convenient, in those cases where a racemic Fmoc-amino acid 11 is sequenced into the polypeptide, the individual stereoisomers are separated during the purification procedure. The Fmoc-amino acids 2, 4, 7, 9, 21, 25 and 26 as well as the acylating agents and isocyanates used to N-cap the polypeptides are known compounds that are commercially available.
The Fmoc-amino acids 10 and 11 are prepared as described herein by methods that are well known to those of ordinary skill in the practice of organic chemistry. In Scheme I, the preparation of Fmoc-amino acids from cyclic ketones is outlined. The 4-phenylcyclohexanones of formula 36 are converted to the hydantoins of formula 37 by treatment with ammonium carbonate and potassium cyanide. The reaction is conveniently carried out an aqueous ethanol mixture at a temperature of from 50xc2x0 C. to 90xc2x0 C., preferably between 80xc2x0 C. and 90xc2x0 C. Direct hydrolysis of the hydantoins to the amino acids of structure 38 require a prolonged treatment with strong base, for example with 6N sodium hydroxide solution or with barium hydroxide at reflux temperature. Alternatively, compounds of structure 37 can be converted to the bis-Boc derivatives of structure 39. The reaction is carried out using tert-butyl dicarbonate [(Boc)2O] in an inert solvent, preferably tetrahydrofuran (THF), in the presence of an organic amine base, preferably TEA and a catalyst, 4-dimethylaminopyridine (DMAP) at a temperature of from zero degrees to room temperature, preferably at room temperature. The bis-Boc hydantoins of structure 39 are readily converted to the amino acids of structure 38. The reaction is accomplished using 1N sodium hydroxide in an inert solvent, preferably dimethoxyethane (DME) at from zero degrees to 50xc2x0 C., preferably at about room temperature. Protection of the amino functionality with an Fmoc group in a compound of structure 38 is carried out under a variety of reaction conditions to give 40. The reaction may conveniently be performed by treatment of a solution of the amino acid 38 in a mixture of THF or dioxane, preferably dioxane and aqueous sodium carbonate with 9-fluorenylmethoxychloroformate (FmocCl) at a temperature of from zero degrees to room temperature, preferably at room temperature. Alternatively, N-(9-fluorenylmethoxycarbonyloxy)succinimide (FmocOSu) is added to a solution of the amino acid 38 in aqueous acetonitrile containing an organic tertiary amine base, preferably TEA. The reaction is run at from zero degrees to room temperature, preferably at room temperature. In another variation of the procedure, DME is evaporated from the hydrolysis mixture in the conversion of 39 to 38 and the reaction is adjusted to xcx9cpH 11. The resulting solution of the sodium salt of 38 is then treated in situ with FmocOSu or FmocCl in dioxane at a temperature of from zero degrees to room temperature, preferably at room temperature.
In the same manner, the tetralones 41, the N-aryl-4-ketopiperidines 42, and the cyclohexanone derivatives 43 and 44 are converted to the corresponding Fmoc-amino acids of structures 11 and 45-47.
Compounds of structure 40 where R3 represents a linear or branched lower alkoxy and R2 and R4 is hydrogen, as in the sub genus structure 49, may be prepared by 0-alkylation of the compound of structure 48 (Scheme J). Where R16 represents an unbranched lower alkyl moiety, the alkylation is carried out by using a primary alkyl halide of structure R16X in the presence of an alkali metal carbonate, for example, sodium or potassium carbonate. The alkyl halide may be a chloro, bromo or iodo derivative, preferably an alkyl iodide (X=I). The reaction may be conveniently carried out in an inert solvent that promotes Sn2 displacement reactions, for example acetone, 2-butanone or N,N-dimethylformamide, preferably acetone, at a temperature of from room temperature to the reflux temperature of the solution, preferably the reflux temperature. When R16 represents a branched lower alkyl group, e.g., 2-propyl, the alkylation is carried out by using a secondary alkyl halide of structure R16X in the presence of an alkali metal carbonate, e.g., potassium carbonate. The secondary alkyl halide is preferably a secondary alkyl iodide, for example, 2-iodopropane (X=I). The reaction may be conveniently carried out in an inert solvent, preferably N,N-dimethylformamide, at a temperature of from room temperature to the reflux temperature of the solution, preferably at about 100xc2x0 C.
Compounds of structure 40 can be prepared by methods that are well known to one of ordinary skill in the practice of organic chemistry. As outlined in Scheme K), treatment of the aryl halides of structure 50 (Xxe2x80x2 represents bromo or iodo) with an alkyl metal reagent, preferably t-butyl lithium, results in a transmetalation reaction to give the corresponding aryl lithium of structure 51. The reaction is conveniently carried out at xe2x88x9278xc2x0 C. by the addition of a solution of the alkyl lithium in to a solution of compounds of structure 50 an inert anhydrous solvent, such as diethyl ether or tetrahydrofuran, preferably tetrahydrofuran. The aryl lithium of structure 51, is then reacted in situ with a solution of the monoketal of cyclohexane-1,4-dione (52) in an suitable inert solvent, for example tetrahydrofuran, while the reaction temperature is maintained below xe2x88x9260xc2x0 C., preferably at about xe2x88x9278xc2x0 C. to give the carbinols of structure 53. The compounds of structure 54 are obtained by the dehydration of the carbinols of structure 53. The reaction is conveniently carried out using a strong organic acid catalyst, preferably p-toluenesulfonic acid in an inert solvent, for example benzene or toluene, preferably benzene, at the reflux temperature of the solvent. The formed water is removed from the reaction mixture by means of a Dean Stark apparatus to enable the reaction to go to completion. Compounds of structure 55 are produced by hydrogenation of the olefins of structure 54. The reaction is conveniently carried out using a noble metal catalyst, for example palladium on carbon, in a hydrogen atmosphere in an inert solvent, for example ethanol or ethyl acetate. The hydrogenation is usually carried out at room temperature and 40 psi of hydrogen, however if the aryl ring in structure 54 contains a group prone to hydrogenolysis, e.g., if R2, R3 or R4 represents chloro, the reaction pressure is kept at about 5 psi. Compounds of structure 55 may be also obtained directly from carbinols of structure 53 by reductive elimination of the hydroxyl group. In this reaction a solution of the compound of structure 53 (R2xe2x95x90R3xe2x95x90H and R4xe2x95x90OMe) in an inert solvent, for example dichloromethane, is treated with a Lewis acid, such as boron trifluoride etherate, and a reducing agent, for example triethylsilane, at a temperature of from zero degrees to room temperature. Removal of the ketal protecting group in compounds of structure 55 gives the ketone of formula 40. The reaction is conveniently carried out in acetone or 2-butanone, preferably acetone under acid catalysis, for example 4N hydrochloric acid or p-toluenesulfonic acid at from room temperature to the reflux temperature of the reaction mixture, preferably at the reflux temperature.
5-Substituted-beta-tetralones of structure 41 are generally known compounds, or if they are not known they can be prepared by methods that are well known to one of ordinary skill in the field of organic chemistry. In the present instance, compounds of structure 41 are prepared by two methods outlined in Schemes L and M.
As shown in Scheme L, a 2-substituted hydrocinnamic acid of structure 56 (R10=bromo, chloro or a linear or branched alkyl group of from 1 to 3 carbons) is converted to the corresponding carboxylic acid chloride of structure 57. This conversion can be carried out by several methods, for example by treatment of the hydrocinnamic acid with oxalyl chloride, optionally in the presence of a catalytic amount of N,N-dimethylformamide, in an inert solvent, such as benzene or dichloromethane, preferably dichloromethane. The reaction may be conveniently carried out at a temperature of from zero degrees to room temperature, preferably at room temperature. Alternatively the compound of structure 56 is reacted with an acyl chloride forming reagent such as sulfuryl chloride in an inert solvent, for example benzene or toluene, preferably toluene at a temperature between room temperature to the reflux temperature of the solution, preferably at the reflux temperature.
The diazoketone of structure 58 is prepared by treatment of the thus formed acyl halide of structure 57 in an inert solvent, e.g., dichloromethane with an excess of a freshly prepared ethereal solution of diazomethane. The combination of reagents is conveniently carried out at ice bath temperature and the reaction is then allowed to proceed at a temperature of from zero degrees to room temperature, preferably at room temperature. Cyclization of the diazoketone of structure 58 to furnish the tetralone of structure 41 is promoted by rhodium (II) acetate dimer in an inert solvent, e.g., dichloromethane. The reaction is normally carried out at from room temperature to the reflux temperature of the solution, preferably at the reflux temperature.
Compounds of structure 41, wherein R10 represents a linear or branched lower alkoxy group or a dialkylamino substituent, are prepared as shown in Scheme M. The compounds of structure 60 (R15xe2x80x2=an unbranched lower alkyl moiety) are prepared by per-O-alkylation of the naphthalenediol of structure 59 with a primary alkyl iodide or bromide, preferably an iodide, in the presence of a base such as an alkali metal carbonate, for example, sodium or potassium carbonate. The reaction may be carried out in an inert solvent, preferably N,N-dimethylformamide at a temperature of from room temperature to 100xc2x0 C., preferably at 35xc2x0 C. The compounds of structure 63 (R15xe2x80x3 represents a branched lower alkyl) are prepared in two steps from the 2-tetralone of structure 61. The tetralone of structure 61 is subjected to dehydrogenation in the presence of a noble metal catalyst, such as palladium metal (10% on carbon) in a suitable high boiling solvent such as p-cymene to give the aromatized compound of structure 62. The naphthol of structure 62 is then 0-alkylated with a secondary alkyl iodide in the presence of a base such as an alkali metal carbonate, preferably cesium carbonate to furnish the compound of structure 63. The reaction may be conveniently carried out in an inert solvent, preferably N,N-dimethylformamide at a temperature of from room temperature to 100xc2x0 C., preferably at about 40xc2x0 C. The compound of structure 65 is prepared by alkylation of 5-amino-2-naphthol (64) with methyl iodide in the presence of a base such as an alkali metal carbonate, preferably potassium carbonate. The reaction may be carried out in an inert solvent, for example acetone or 2-butanone, preferably acetone, at a temperature between room temperature and the reflux temperature of the solution, preferably at the reflux temperature.
The tetralones of structures 41 are produced by reduction of the compounds of structures 60, 63 and 65 under dissolving metal conditions, followed by the acid catalyzed hydrolysis of the intermediate enol ethers. The transformation is conveniently carried out by the portionwise addition of a large excess of an alkali metal, such as sodium or potassium, preferably sodium, to a boiling solution of the substrate in an lower alcohol, preferably alcohol until the starting material is consumed. The tetralones of structures 41 are obtained by treatment of a solution of the isolated intermediate enol ethers with a strong acid catalyst, preferably p-toluenesulfonic acid. The hydrolysis may conveniently carried out in a mixture of a lower alcohol, preferably ethanol, and water at a temperature of between room temperature and the reflux temperature of the solution, preferably at the reflux temperature.
Compounds of structure 68 are can be prepared by reactions that are known per se. For example, they can be prepared by coupling a secondary amine of structure 66 with an aryl bromide or iodide, preferably an aryl iodide of structure 67 (Scheme N). The coupling reaction is catalyzed by a noble metal catalyst, preferably tri(dibenzylideneacetone)-dipalladium, in the presence of a chelating phosphine ligand, preferably tri-o-tolylphosphine, and a hindered alkoxide base such as sodium tert-butoxide. The reaction is conveniently carried out in an inert atmosphere using an anhydrous solvent such as dioxane or toluene, preferably dioxane, at a temperature of from 60xc2x0 C. to the reflux temperature, preferably at 90xc2x0 C. Compounds of structure 56 and 66 are generally known compounds and are can be obtained from commercial sources. Removal of the carbonyl protecting group in compound 67 to give compounds of structure 42 can be carried out by a variety of methods well known in the field of organic chemistry. For example, the deprotection can be achieved by treatment of a solution of compound 68 in a low boiling ketone such as acetone or 2-butanone with an aqueous mineral acid solution, for example 6N hydrochloric acid. The reaction can be run at a temperature of from room temperature to the reflux temperature of the mixture, preferably at the reflux temperature.
The cyclohexanone derivatives of structures 63 are commercially available compounds and the 4,4-diphenylcyclohexanone (64) is prepared by published procedures.
This invention will be better understood by reference to the following examples, which illustrate but do not limit the invention described herein.